Proteases with improved enzyme stability in detergents

ABSTRACT

The present disclosure relates to proteases having an amino acid sequence with at least 70% sequence identity to the amino acid sequence given in SEQ ID No. 1, across its whole length, and comprising an amino acid substitution on at least one of the positions P9, Q10, Q62, L82, P86, N130, T141, N187, S236 or T253, relating in each case to the numbering according to SEQ ID No. 1. The present disclosure also relates to the production and use thereof. Said type of proteases have a very good cleaning performance.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. National-Stage entry under 35 U.S.C. § 371based on International Application No PCT/EP2017/055276, filed Mar. 7,2017 which was published under PCT Article 21(2) and which claimspriority to German Application No. 10 2016 204 814.7, filed Mar. 23,2016, which are all hereby incorporated in their entirety by reference.

TECHNICAL FIELD

The present disclosure lies in the field of enzyme technology. Thepresent disclosure relates to proteases from Bacillus pumilus of whichthe amino acid sequence has been altered, in particular with respect touse in washing and cleaning agents, in order to impart improved cleaningperformance thereto, to the nucleic acids which code for said proteases,and to the preparation of said proteases. The present disclosure furtherrelates to uses of said proteases, to methods in which said proteasesare used, and to agents, in particular washing and cleaning agents,which contain said proteases.

BACKGROUND

Proteases are among the industrially most significant enzymes of all. Inthe context of washing and cleaning agents, proteases are the longestestablished enzymes that are contained in virtually all modern,high-performance washing and cleaning agents. They cause the breakdownof protein-containing stains on the item to be cleaned. Of theseproteases, subtilisin-type proteases (subtilases, subtilopeptidases, EC3.4.21.62) are particularly significant, which proteases are serineproteases owing to the catalytically active amino acids. Said proteasesact as non-specific endopeptidases and hydrolyze any acid amide bondsthat are within peptides or proteins. Their pH optimum is usually in thehighly alkaline range. An overview of this family is found, for example,in the article “Subtilases: Subtilisin-like Proteases” by R. Siezen,pages 75-95 in “Subtilisin enzymes”, published by R. Bott and C. Betzel,New York, 1996. Subtilases are formed naturally by microorganisms. Ofthese, subtilisins that are formed by and secreted from Bacillus speciesshould be mentioned in particular as the most significant group withinthe subtilases.

Examples of the subtilisin-type proteases that are preferably used inwashing and cleaning agents are the subtilisins BPN' and Carlsberg,protease PB92, subtilisins 147 and 309, protease from Bacillus lentus,in particular from Bacillus lentus DSM 5483, subtilisin DY, the enzymesthermitase, proteinase K and proteases TW3 and TW7, which belong to thesubtilases but no longer to the subtilisins in the narrower sense, andvariants of the mentioned proteases which have an altered amino acidsequence by comparison with the starting protease. Proteases arealtered, selectively or randomly, by methods known from the prior art,and are thereby optimized for use in washing and cleaning agents, forexample. These methods include point, deletion or insertion mutagenesis,or fusion with other proteins or protein parts. Therefore, variants thatare appropriately optimized are known for most of the proteases knownfrom the prior art.

European patent application EP 2016175 A1 discloses, for example, aprotease from Bacillus pumilus provided for washing and cleaning agents.Generally, only select proteases are in any way suitable for use inliquid surfactant-containing preparations. Several proteases do not havesufficiently high levels of catalytic performance in preparations ofthis kind. Therefore, when using proteases in cleaning agents, it isparticularly desirable for said proteases to have a high level ofcatalytic activity under conditions that prevail during a washingprocess.

As a result, protease-containing and surfactant-containing liquidformulations from the prior art are disadvantageous in that thecontained proteases do not have a satisfactory level of proteolyticactivity under standard washing conditions, and therefore theformulations do not have optimum cleaning performance onprotease-sensitive stains.

BRIEF SUMMARY

A protease having an amino acid sequence is provided herein. Theprotease has an at least about 70% sequence identity to the amino acidsequence set forth in SEQ ID NO:1 over the entire length thereof. Theprotease has an amino acid substitution at one or more of positions P9,Q10, Q62, L82, P86, N130, T141, N187, S236 or T253, each based on thenumbering according to SEQ ID NO:1.

A method for preparing a protease is also provided herein. The methodincludes substituting an amino acid at one or more of the positionswhich correspond to positions 9, 10, 62, 82, 86, 130, 141, 187 and 236in SEQ ID NO:1 in a starting protease of which the sequence is at leastabout 70% identical to the amino acid sequence shown in SEQ ID NO:1 overthe entire length thereof amino acid.

DETAILED DESCRIPTION

The following detailed description is merely exemplary in nature and isnot intended to limit the disclosure or the application and uses of thesubject matter as described herein. Furthermore, there is no intentionto be bound by any theory presented in the preceding background or thefollowing detailed description.

It has surprisingly now been found that a protease from Bacillus pumilusor a reasonably similar protease (in terms of sequence identity) whichhas an amino acid substitution at at least one of positions P9, Q10,Q62, L82, P86, N130, T141, N187, S236 or T253, in each case based on thenumbering according to SEQ ID NO:1, is improved in terms of proteolyticactivity under standard washing conditions by comparison with thewild-type form, and therefore is particularly suitable for use inwashing or cleaning agents.

Therefore, in a first aspect, the present disclosure relates to aprotease having an amino acid sequence which is at least about 70%identical to the amino acid sequence shown in SEQ ID NO:1 over theentire length thereof, and has an amino acid substitution at at leastone of positions P9, Q10, Q62, L82, P86, N130, T141, N187, S236 or T253,in each case based on the numbering according to SEQ ID NO:1.

The present disclosure further relates to a method for preparing aprotease, comprising substituting an amino acid at at least one positionwhich corresponds to position 9, 10, 62, 82, 86, 130, 141, 187 or 236 inSEQ ID NO:1 in a starting protease of which the sequence is at leastabout 70% identical to the amino acid sequence shown in SEQ ID NO:1 overthe entire length thereof, preferably such that the protease has atleast one of the amino acid substitutions P9H, Q10E, Q62E, L82F, P86S,P86A, N130D, T141K, N187H, S236A or T253S.

A “protease” within the meaning of the present patent applicationtherefore covers both the protease as such and a protease prepared usinga method as contemplated herein. All comments made with regard to theprotease therefore relate to both the protease as such and the proteasesprepared using corresponding methods.

Other aspects of the present disclosure relate to the nucleic acidswhich code for these proteases, to non-human host cells which containproteases or nucleic acids as contemplated herein, to agents, inparticular washing and cleaning agents, which comprise proteases ascontemplated herein, to washing and cleaning methods, and to uses of theproteases as contemplated herein in washing or cleaning agents forremoving fat-containing stains.

“At least one”, as used herein, means one or more, i.e. one, two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, or more.

The present disclosure is based on the surprising finding of theinventor that an amino acid substitution at at least one of positions 9,10, 62, 82, 86, 130, 141, 187 or 236 of the protease from Bacilluspumilus according to SEQ ID NO:1 in a protease which has an amino acidsequence that is at least about 70% identical to the amino acid sequenceshown in SEQ ID NO:1 such that the amino acids 9H, 10E, 62E, 82F, 86S,86A, 130D, 141K, 187H, 236A or 253S are present at at least one of thecorresponding positions results in improved catalytic activity of saidaltered protease in washing and cleaning agents. This is particularlysurprising since none of the above-mentioned amino acid substitutionshas previously been associated with increased catalytic activity of theprotease.

The proteases as contemplated herein have increased catalytic activityin washing or cleaning agents. In a wide variety of embodiments, theproteases as contemplated herein have a proteolytic activity of at leastabout 110%, at least about 115%, at least about 120%, at least about125%, at least about 130%, at least about 135%, at least about 140%, atleast about 145%, at least about 150%, at least about 155% or at leastabout 160%, based on the wild-type variant of the protease (SEQ IDNO:1). Proteases of this kind that are improved in terms of performanceprovide for improved washing results on proteolytically sensitive stainsin a wide temperature range.

The proteases as contemplated herein have enzymatic activity, i.e. theyare capable of hydrolyzing peptides and proteins, in particular in awashing or cleaning agent. A protease as contemplated herein istherefore an enzyme which catalyzes the hydrolysis of amide/peptidebonds in protein/peptide substrates and is therefore capable of cleavingproteins or peptides. Furthermore, a protease as contemplated herein ispreferably a mature protease, i.e. the catalytically active moleculewithout signal peptide(s) and/or propeptide(s). Unless indicatedotherwise, the specified sequences also each relate to mature(processed) enzymes.

In various embodiments, the protease as contemplated herein contains atleast one amino acid substitution selected from the group including P9H,Q10E, Q62E, L82F, P86S, P86A, N130D, T141K, N187H, S236A or T253S, ineach case based on the numbering according to SEQ ID NO:1. In morepreferred embodiments, the protease as contemplated herein contains oneof the following amino acid-substitution variants: (i) P86S and S236A;(ii) Q62E; (iii) Q62E and L82F; (iv) Q10E; (v) N130D, T141K and T253S;(vi) P86A; (vii) P9H, Q62E and L82F; (viii) Q62E, L82F and N187H; (ix)Q62E and N130D; or (x) P86S, N187H and S236A, the numbering being basedin each case on the numbering according to SEQ ID NO:1.

In a further embodiment of the present disclosure, the protease has anamino acid sequence that is at least about 70%, about 71%, about 72%,about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 90.5%,about 91%, about 91.5%, about 92%, about 92.5%, about 93%, about 93.5%,about 94%, about 94.5%, about 95%, about 95.5%, about 96%, about 96.5%,about 97%, about 97.5%, about 98%, about 98.5% and about 98.8% identicalto the amino acid sequence shown in SEQ ID NO:1 over the entire lengththereof, and has one or more of the amino acid substitutions 9H, 10E,62E, 82F, 86S, 86A, 130D, 141K, 187H, 236A or 253S at at least one ofpositions 9, 10, 62, 82, 86, 130, 141, 187 or 236 in the numberingaccording to SEQ ID NO:1. In the context of the present disclosure, thefeature whereby a protease has the stated substitutions means that itcontains at least one of the corresponding amino acids at thecorresponding positions, i.e. not all of the ten positions are otherwisemutated or deleted, for example by fragmentation of the protease. Theamino acid sequences of proteases of this kind that are preferred ascontemplated herein are shown in SEQ ID NOs:2-11.

The identity of nucleic-acid or amino acid sequences is determined by asequence comparison. This sequence comparison is based on the BLASTalgorithm (cf. for example Altschul, S. F., Gish, W., Miller, W., Myers,E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J.Mol. Biol. 215:403-410 and Altschul, Stephan F., Thomas L. Madden,Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, andDavid J. Lipman (1997): “Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs”; Nucleic Acids Res., 25, pages3389-3402) which is established and commonly used in the prior art andis carried out, in principle, by similar series of nucleotides or aminoacids in the nucleic-acid or amino acid sequences being assigned to oneanother. The assignment of the relevant positions shown in a table isreferred to as an “alignment”. Another algorithm available from theprior art is the FASTA algorithm. Sequence comparisons (alignments), inparticular multiple sequence comparisons, are generated using computerprograms. For example, the Clustal series (cf. for example Chenna et al.(2003): Multiple sequence alignment with the Clustal series of programs.Nucleic Acid Research 31, 3497-3500), T-Coffee (cf. for exampleNotredame et al. (2000): T-Coffee: A novel method for multiple sequencealignments. J. Mol. Biol. 302, 205-217) or programs based on theseprograms or algorithms are often used. Sequence comparisons (alignments)are also possible that use the computer program Vector NTI® Suite 10.3(Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, Calif., USA)with the specified standard parameters, the AlignX-Modul of whichprogram for the sequence comparisons is based on ClustalW. Unlessindicated otherwise, sequence identity specified herein is determinedusing the BLAST algorithm.

A comparison of this kind makes it possible to specify the similaritybetween the compared sequences. This similarity is usually expressed inpercent identity, i.e. the percentage of identical nucleotides or aminoacid residues at the same positions or at positions that correspond toone another in an alignment. In amino acid sequences, the broaderconcept of “homology” factors in conserved amino acid exchanges, i.e.amino acids having similar chemical activity, since these usually havesimilar chemical activities within the protein. Therefore, thesimilarity between the compared sequences can also be expressed inpercent homology or percent similarity. Information relating to identityand/or homology may apply to the entirety of the polypeptides or genesor only to individual segments. Homologous or identical segments ofdifferent nucleic-acid or amino acid sequences are therefore defined bymatches in the sequences. Segments of this kind often have identicalfunctions. Said segments may be small and only comprise a fewnucleotides or amino acids. Segments that are this small often performfunctions that are essential to the overall activity of the protein.Therefore, it may be expedient for sequence matches to only relate toindividual, optionally small, segments. However, unless indicatedotherwise, information relating to identity or homology in the presentapplication relates to the entire length of the nucleic-acid or aminoacid sequence specified in each case.

In the context of the present disclosure, if it is stated that an aminoacid position corresponds to a numerically identified position in SEQ IDNO:1, this means that the corresponding position is assigned to thenumerically identified position in SEQ ID NO:1 in an alignment asdefined above.

In a further embodiment of the present disclosure, the protease cleaningperformance is not significantly reduced by comparison with that of aprotease which has an amino acid sequence that corresponds to the aminoacid sequence shown in SEQ ID NO:1, i.e. said protease has at leastabout 80%, preferably at least about 100%, more preferably at leastabout 110% or more, of the reference washing performance. The cleaningperformance can be determined in a washing system which contains awashing agent in a dosage of between from about 4.5 and about 7.0 gramsper liter of washing liquor, and the protease, the proteases to becompared being used in the same concentration (based on the activeprotein) and the cleaning performance with respect to a stain on cottonis determined by measuring the extent to which the washed textiles havebeen cleaned. For example, the washing process can be carried out forabout 60 minutes at a temperature of about 40° C., and the water canhave a water hardness of between from about 15.5 and about 16.5° (Germandegree of hardness). The concentration of the protease in the washingagent intended for this washing system is from about 0.001 to about 0.1wt. %, preferably from about 0.01 to about 0.06 wt. %, based on thepurified active protein.

A liquid reference washing agent for a washing system of this kind canbe composed as follows (all amounts are given in percent by weight):about 4.4% of alkyl benzene sulfonic acid, about 5.6% of further anionicsurfactants, about 2.4% of C12-C18 Na salts of fatty acids (soaps),about 4.4% of non-ionic surfactants, about 0.2% of phosphonates, about1.4% of citric acid, about 0.95% of NaOH, about 0.01% of defoamers,about 2% of glycerol, about 0.08% of preservatives, about 1% of ethanol,and the remaining percentage of demineralized water. The dosage of theliquid washing agent is preferably between from about 4.5 and about 6.0grams per liter of washing liquor, for example about 4.7, about 4.9 orabout 5.9 grams per liter of washing liquor. Washing is preferablycarried out within a pH range of between from about pH 8 and about pH10.5, preferably between from about pH 8 and about pH 9.

Within the scope of the present disclosure, the cleaning performance isdetermined, for example, at about 40° C. using a liquid washing agent asspecified above, the washing process preferably being carried out forabout 60 minutes at about 600 rpm.

The degree of whiteness, i.e. the lightening of the stains, isdetermined using optical measurement methods, preferablyphotometrically, as a measure of cleaning performance. A device suitablefor this purpose is the spectrometer Minolta CM508d, for example. Thedevices used for the measurement are usually calibrated, in advance,against a white standard, preferably a white standard that is suppliedtherewith.

Each protease being applied in an identical manner in terms of activityensures that the relevant enzymatic properties, i.e. for examplecleaning performance on particular stains, are compared even if there issome kind of divergence in the ratio of active substance to overallprotein (the values for specific activity). In general, low specificactivity can be compensated for by adding a larger amount of protein.

In other respects, a person skilled in the art of enzyme technology isfamiliar with methods for determining protease activity, and he usessaid methods as a matter of routine. For example, methods of this kindare disclosed in Tenside, Band 7 (1970), pages 125-132. Alternatively,protease activity can be determined by releasing the chromophorepara-nitroaniline (pNA) from the substratesuc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF). The protease cleavesthe substrate and releases pNA. The release of pNA causes an increase inthe extinction at about 410 nm, the temporal progression of which is ameasure of enzymatic activity (cf. Del Mar et al., 1979). Themeasurement is taken at a temperature of about 25° C., a pH of about8.6, and a wavelength of about 410 nm. The measurement time is about 5min and the measurement interval is from about 20 s to about 60 s.Protease activity is usually expressed in protease units (PU). Suitableprotease activities are, for example, 2.25, 5 or 10 PU per ml of washingliquor. However, the protease activity is not zero.

An alternative test for establishing the proteolytic activity of theproteases as contemplated herein is an optical measurement method,preferably a photometric method. The test suitable for this purposecomprises the protease-dependent cleavage of the substrate proteincasein. Said protein is cleaved by the protease into a plurality ofsmaller subproducts. All of these subproducts have an increasedabsorption at about 290 nm by comparison with uncleaved casein, thisincreased absorption being determined using a photometer, and it thusbeing possible to draw a conclusion on the enzymatic activity of theprotease.

The protein concentration can be determined using known methods, forexample the BCA method (bicinchoninic acid;2,2′-biquinolyl-4,4′-dicarboxylic acid) or the Biuret method (A. G.Gornall, C. S. Bardawill and M. M. David, J. Biol. Chem., 177 (1948),pages 751-766). The active protein concentration can be determined, inthis respect, by titrating the active centers using a suitableirreversible inhibitor, and determining the residual activity (cf. M.Bender et al., J. Am. Chem. Soc. 88, 24 (1966), pages 5890-5913).

In addition to the aforementioned amino acid alterations, proteases ascontemplated herein can have further amino acid alterations, inparticular amino acid substitutions, insertions or deletions. Proteasesof this kind are developed, for example, by targeted genetic alteration,i.e. by mutagenesis methods, and optimized for particular uses or inrespect of specific properties (for example in respect of theircatalytic activity, stability, etc.). Furthermore, nucleic acids ascontemplated herein can be incorporated in recombination approaches andare thus used to produce completely new types of proteases or otherpolypeptides.

The aim is to introduce targeted mutations, such as substitutions,insertions or deletions, into known molecules, in order to improve thecleaning performance of enzymes as contemplated herein, for example. Forthis purpose, in particular the surface charges and/or isoelectric pointof the molecules, and thus their interactions with the substrate, can bealtered. For example, the net charge of the enzymes can be changed inorder to thereby influence the substrate binding, in particular for usein washing and cleaning agents. Alternatively or in addition, thestability or catalytic activity of the protease can be increased, andthus the cleaning performance thereof can be improved, by one or moreappropriate mutations. Advantageous properties of individual mutations,e.g. individual substitutions, may complement one another. A proteasethat has already been optimized in terms of particular properties, forexample in terms of the stability thereof during storage, can thereforealso be developed within the scope of the present disclosure.

In order to describe substitutions that affect exactly one amino acidposition (amino acid exchanges), the following convention is used: theinternationally conventional single-letter code of the naturally presentamino acid is given first, and then the associated sequence position,and finally the amino acid that has been added. Several exchanges withinthe same polypeptide chain are separated from one another by slashes.For insertions, additional amino acids are indicated after the sequenceposition. For deletions, the amino acid that has been removed isreplaced with a symbol, for example a star or a dash, or a Δ is putbefore the corresponding position. For example, P9H denotes thesubstitution of proline at position 9 by histidine, P9HT denotes theinsertion of threonine after the amino acid histidine at position 9, andP9* or ΔP9 denotes the deletion of proline from position 9. Thisnomenclature is known to a person skilled in the art of enzymetechnology.

Therefore, the present disclosure further relates to a protease whichcan be obtained from a protease as described above acting as a startingmolecule by employing one or more conservative amino acid substitutions,the protease still having in the numbering according to SEQ ID NO:1 atleast one of the amino acid substitutions as contemplated herein at thepositions which correspond to positions 9, 10, 62, 82, 86, 130, 141, 187and 236 in SEQ ID NO:1, as described above. The term “conservative aminoacid substitutions” means the exchange (substitution) of an amino acidresidue with another amino acid residue, this exchange not resulting ina change in the polarity or charge at the position of the exchangedamino acid, e.g. the exchange of a nonpolar amino acid residue withanother nonpolar amino acid residue. Within the scope of the presentdisclosure, conservative amino acid substitutions include for example:G=A=S, l=V=L=M, D=E, N=Q, K=R, Y=F, S=T, G=A=I=V=L=M=Y=F=W=P=S=T.

Alternatively or in addition, the protease can be obtained from aprotease as contemplated herein acting as a starting molecule byemploying fragmentation, deletion, insertion or substitution mutagenesisand has an amino acid sequence which matches that of the startingmolecule over a length of at least about 200, about 210, about 220,about 230, about 240, about 250, about 260, about 270 or about 275interconnected amino acids, the amino acid substitution(s) contained inthe starting molecule still being present at one or more of thepositions which correspond to positions 9, 10, 62, 82, 86, 130, 141, 187and 236 in SEQ ID NO:1.

It is thus possible, for example, for individual amino acids to bedeleted from the enzyme termini or loops, without this resulting in theproteolytic activity being lost or reduced. Furthermore, by employingfragmentation, deletion, insertion or substitution mutagenesis of thiskind, the allergenicity of relevant enzymes, for example, can also bereduced and thus the usability thereof can be improved overall. Theenzymes advantageously still have their proteolytic activity even afterthe mutagenesis, i.e. the proteolytic activity thereof corresponds atleast to that of the starting enzyme, i.e. in a preferred embodiment,the proteolytic activity is at least about 80%, preferably at leastabout 90%, of the activity of the starting enzyme. Other substitutionscan also have advantageous effects. It is possible to exchange a singleamino acid or several interconnected amino acids with other amino acids.

Alternatively or in addition, the protease can be obtained from aprotease as contemplated herein acting as a starting molecule byemploying one or more conservative amino acid substitutions, theprotease having at least one of the amino acid substitutions P9H, Q10E,Q62E, L82F, P86S, P86A, N130D, T141K, N187H, S236A or T253S at thepositions which correspond to positions 9, 10, 62, 82, 86, 130, 141, 187and 236 according to SEQ ID NO:1.

In further embodiments, the protease can be obtained from a protease ascontemplated herein acting as a starting molecule by employingfragmentation, deletion, insertion or substitution mutagenesis, and hasan amino acid sequence which matches that of the starting molecule overa length of at least about 200, about 210, about 220, about 230, about240, about 250, about 260, about 270 or about 275 interconnected aminoacids, the protease having at least one of the amino acid substitutionsP9H, Q10E, Q62E, L82F, P86S, P86A, N130D, T141K, N187H, S236A or T253Sat the positions which correspond to positions 9, 10, 62, 82, 86, 130,141, 187 and 236 according to SEQ ID NO:1.

In this case, the other amino acid positions are defined by an alignmentof the amino acid sequence of a protease as contemplated herein with theamino acid sequence of the protease from Bacillus pumilus, as shown inSEQ ID NO:1. Furthermore, the assignment of the positions is determinedby the mature protein. In particular, this assignment is also used ifthe amino acid sequence of a protease as contemplated herein has ahigher number of amino acid residues than the protease from Bacilluspumilus according to SEQ ID NO:1. Proceeding from the mentionedpositions in the amino acid sequence of the protease from Bacilluspumilus, the alteration positions in a protease as contemplated hereinare those which are precisely assigned to these positions in analignment.

Advantageous positions for sequence alterations, in particularsubstitutions, of the protease from Bacillus pumilus which whentransferred to homologous positions of the proteases as contemplatedherein are preferably of significance and impart advantageous functionalproperties to the protease are therefore the positions which correspondto positions 9, 10, 62, 82, 86, 130, 141, 187 and 236 in SEQ ID NO:1 inan alignment, i.e. in the numbering according to SEQ ID NO:1. At thestated positions, the following amino acid residues are present in thewild-type molecule of the protease from Bacillus pumilus: P9, Q10, Q62,L82, P86, N130, T141, N187, S236 and T253.

Further confirmation of the correct assignment of the amino acids to bealtered, i.e. in particular of the functional correspondence thereof,can be provided by comparison tests during which the two positionsassigned to one another on the basis of an alignment are altered in thesame way in the two proteases being compared with one another and it isobserved whether the enzymatic activity is altered in the same way inthe two proteases. If, for example, an amino acid exchange at aparticular position of the protease from Bacillus pumilus according toSEQ ID NO:1 is associated with a change in an enzymatic parameter, forexample with the increase in the K_(M) value, and if a correspondingchange in the enzymatic parameter, thus for example also an increase inthe K_(M) value, is observed in a protease variant as contemplatedherein of which the amino acid exchange was achieved by the same addedamino acid, this is considered to be confirmation of the correctassignment.

All elements specified can also be applied to the methods ascontemplated herein for preparing a protease. A method as contemplatedherein therefore further comprises one or more of the following methodsteps:

a) introducing one or more conservative amino acid substitutions, theprotease having at least one of the amino acid substitutions P9H, Q10E,Q62E, L82F, P86S, P86A, N130D, T141K, N187H, S236A or T253S at thepositions which correspond to positions 9, 10, 62, 82, 86, 130, 141, 187and 236 according to SEQ ID NO:1;b) altering the amino acid sequence by employing fragmentation,deletion, insertion or substitution mutagenesis such that the proteasehas an amino acid sequence which matches that of the starting moleculeover a length of at least about 200, about 210, about 220, about 230,about 240, about 250, about 260, about 270 or about 275 interconnectedamino acids, the protease having at least one of the amino acidsubstitutions P9H, Q10E, Q62E, L82F, P86S, P86A, N130D, T141K, N187H,S236A or T253S at the positions which correspond to positions 9, 10, 62,82, 86, 130, 141, 187 and 236 according to SEQ ID NO:1.

All comments made also apply to the methods as contemplated herein.

In further embodiments of the present disclosure, the protease or theprotease prepared using a method as contemplated herein is still atleast about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 90.5%, about 91%, about 91.5%, about 92%,about 92.5%, about 93%, about 93.5%, about 94%, about 94.5%, about 95%,about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%,about 98.5% and about 98.8% identical to the amino acid sequence shownin SEQ ID NO:1 over the entire length thereof. Alternatively, theprotease or the protease prepared using a method as contemplated hereinis still at least about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 90.5%, about 91%, about 91.5%,about 92%, about 92.5%, about 93%, about 93.5%, about 94%, about 94.5%,about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%,or about 98% identical to one of the amino acid sequences shown in SEQID NOs:2-11 over the entire length thereof. The protease or the proteaseprepared using a method as contemplated herein has an amino acidsubstitution at at least one of positions P9, Q10, Q62, L82, P86, N130,T141, N187, S236 or T253, in each case based on the numbering accordingto SEQ ID NO:1. In more preferred embodiments, the amino acidsubstitution is at least one substitution selected from the groupincluding P9H, Q10E, Q62E, L82F, P86S, P86A, N130D, T141K, N187H, S236Aand T253S, in each case based on the numbering according to SEQ ID NO:1.In even more preferred embodiments, the protease has one of thefollowing amino acid-substitution variants: (i) P86S and S236A; (ii)Q62E; (iii) Q62E and L82F; (iv) Q10E; (v) N130D, T141K and T253S; (vi)P86A; (vii) P9H, Q62E and L82F; (viii) Q62E, L82F and N187H; (ix) Q62Eand N130D; or (x) P86S, N187H and S236A.

The present disclosure also relates to a protease as described abovewhich is additionally stabilized, in particular by employing one or moremutations, for example substitutions, or by being coupled to a polymer.Increasing stability during storage and/or during use, for exampleduring the washing process, leads to the enzymatic activity beingmaintained for longer and thus to the cleaning performance beingimproved. In principle, all stabilizing possibilities that are expedientand/or described in the prior art can be considered for this.Stabilizations which are achieved by employing mutations of the enzymeitself are preferred, since stabilizations of this kind do not requireany further working steps after the enzyme has been obtained. Examplesof sequence alterations suitable for this purpose have been mentionedabove. Further suitable sequence alterations are known from the priorart.

Further possibilities for stabilization include for example:

-   -   altering the binding of metal ions, in particular the calcium        binding sites, for example by exchanging one or more of the        amino acid(s) that are involved in the calcium binding with one        or more negatively charged amino acids and/or by introducing        sequence alterations in at least one of the sequences of the two        amino acids arginine and glycine;    -   protecting against the influence of denaturing agents, such as        surfactants, by employing mutations which cause the amino acid        sequence to be altered on or at the surface of the protein;    -   exchanging amino acids which are close to the N-terminus with        amino acids which are assumed to come into contact with the rest        of the molecule by employing non-covalent interactions, and thus        contribute to maintaining the globular structure.

Preferred embodiments are those in which the enzyme is stabilized inseveral different ways, since several stabilizing mutations have acumulative or synergistic effect.

The present disclosure also relates to a protease as described abovewhich has at least one chemical modification. A protease that is alteredin this way is referred to as a “derivative”, i.e. the protease isderivatized.

Within the meaning of the present application, “derivatives” aretherefore understood to mean proteins of which the pure amino acid chainhas been modified chemically. Derivatizations of this kind can becarried out in vivo, for example, by the host cell which expresses theprotein. In this respect, couplings to low-molecular-weight compounds,such as lipids or oligosaccharides, are of particular importance.However, derivatizations may also be carried out in vitro, for exampleby the chemical conversion of a side chain of an amino acid or bycovalent bonding of another compound to the protein. For example, it ispossible to couple amines to carboxyl groups of an enzyme in order tochange the isoelectric point. This other compound may also be anotherprotein which is bound to a protein as contemplated herein viabifunctional chemical compounds, for example. Derivatization is alsounderstood to mean covalent bonding to a macromolecular carrier, ornon-covalent inclusion in suitable macromolecular cage structures.Derivatizations can, for example, influence the substrate specificity orthe bond strength to the substrate or cause temporary inhibition ofenzymatic activity, if the coupled substance is an inhibitor. This canbe expedient in terms of the period of storage, for example.Modifications of this kind can also influence stability or enzymaticactivity. They can also be used to reduce the allergenicity and/orimmunogenicity of the protein and to thus increase the skincompatibility thereof, for example. For example, couplings tomacromolecular compounds, for example polyethylene glycol, can improvethe protein in terms of stability and/or skin compatibility.

In the broadest sense, derivatives of a protein as contemplated hereincan be understood to also include preparations of these proteins.Depending on how a protein is obtained, recovered or prepared, saidprotein can be combined with a wide range of other substances, forexample from the culture of the microorganisms that produce it. Aprotein may also have been deliberately mixed with other substances inorder to increase its storage stability, for example. Therefore, thepresent disclosure also covers all preparations of a protein ascontemplated herein. This is still true irrespective of whether or notthis enzymatic activity actually develops in a particular preparation.This is because it may be desirable for the protein to not have anyactivity or to only have low activity when being stored, and for theenzymatic function to only develop once the protein is in use. This canbe controlled, for example, by appropriate accompanying substances. Inparticular, in this respect, it is possible to jointly prepare proteasesand specific inhibitors.

Of all the above-described proteases or protease variants and/orderivatives, within the scope of the present disclosure, proteases,protease variants and/or derivatives of which the catalytic activitycorresponds to that of at least one of the proteases according to SEQ IDNOs:2-11 and/or of which the cleaning performance corresponds to that ofat least one of the proteases according to SEQ ID NOs:2-11 areparticularly preferred, the cleaning performance being determined in awashing system, as described above.

The present disclosure also relates to a nucleic acid which codes for aprotease as contemplated herein, and to a vector containing a nucleicacid of this kind, in particular a cloning vector or an expressionvector.

These may be DNA or RNA molecules. They may be present as a singlestrand, as a single strand that is complementary to the first singlestrand, or as a double strand. In the case of DNA molecules inparticular, the sequences of the two complementary strands should betaken into account in all three possible reading frames. It should alsobe noted that different codons, i.e. base triplets, can code for thesame amino acids, such that a particular amino acid sequence can becoded for by several different nucleic acids. Owing to this degeneracyof the genetic code, all nucleic-acid sequences which can code for oneof the above-described proteases are included in this subject of thepresent disclosure. A person skilled in the art is able to identifythese nucleic-acid sequences with absolute certainty since, despite thedegeneracy of the genetic code, defined amino acids can be assigned toindividual codons. Therefore, proceeding from an amino acid sequence, aperson skilled in the art can easily identify nucleic acids which codefor said amino acid sequence. Furthermore, in nucleic acids ascontemplated herein, one or more codons can be replaced by synonymouscodons. This aspect relates in particular to the heterologous expressionof the enzymes as contemplated herein. Therefore, each organism, forexample a host cell of a production strain, has a particular codonusage. “Codon usage” is understood to mean the translation of thegenetic code into amino acids by employing the relevant organism.Bottlenecks can occur in protein biosynthesis if the codons on thenucleic acid are accompanied by a comparatively low number of chargedtRNA molecules in the organism. Although coding for the same amino acid,this leads to a codon being translated less efficiently in the organismthan a synonymous codon which codes for the same amino acid. Owing tothe presence of a higher number of tRNA molecules for the synonymouscodon, said codon can be translated more efficiently in the organism.

Using methods which are currently generally known, such as chemicalsynthesis or polymerase chain reaction (PCR), in conjunction withstandard methods in molecular biology and/or protein chemistry, it ispossible for a person skilled in the art, on the basis of known DNAand/or amino acid sequences, to produce the corresponding nucleic acidsand even complete genes. Methods of this kind are known from, forexample, Sambrook, J., Fritsch, E. F. and Maniatis, T. 2001. Molecularcloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press.

Within the meaning of the present disclosure, vectors are understood tomean elements which consist of nucleic acids and which contain a nucleicacid as contemplated herein as a characterizing nucleic-acid range.Vectors allow establishment of this nucleic acid in a species or a cellline over multiple generations or cell divisions as a stable geneticelement. Vectors are specific plasmids, i.e. circular genetic elements,in particular for use in bacteria. Within the scope of the presentdisclosure, a nucleic acid as contemplated herein is cloned in a vector.These may include vectors, for example, which originate from bacterialplasmids, from viruses, or from bacteriophages, or predominantlysynthetic vectors or plasmids having elements of various origins. Usingthe further genetic elements which are present in each case, vectors areable to become established as stable units in the host cells in questionover several generations. They may be present as separate units outsideof a chromosome or be integrated in a chromosome or chromosomal DNA.

Expression vectors have nucleic-acid sequences which enable them toreplicate in the host cells, preferably microorganisms, particularlypreferably bacteria, which contain them and to express therein acontained nucleic acid. The expression is influenced, in particular, bypromoter(s) which regulate the transcription. In principle, theexpression can be carried out by the natural promoter which isoriginally located in front of the nucleic acid to be expressed, by apromoter of the host cell provided on the expression vector, or by amodified or completely different promoter of another organism or anotherhost cell. In the present case, at least one promoter is provided forthe expression of a nucleic acid as contemplated herein and is used forthe expression thereof. Expression vectors can also be regulated, forexample by changing the culturing conditions, by reaching a particularcell density in the host cells containing said vectors, or by addingparticular substances, in particular activators for gene expression. Anexample of a substance of this kind is the galactose derivativeisopropyl-ß-D-thiogalactopyranoside (IPTG) which is used as an activatorfor the bacterial lactose operon (lac operon). Unlike in expressionvectors, the contained nucleic acid in cloning vectors is not expressed.

The present disclosure also relates to a non-human host cell containinga nucleic acid as contemplated herein or a vector as contemplatedherein, or containing a protease as contemplated herein, in particular anon-human host cell which secretes the protease into the mediumsurrounding the host cell. A nucleic acid as contemplated herein or avector as contemplated herein is preferably transformed into amicroorganism which then constitutes a host cell as contemplated herein.Alternatively, individual components, i.e. nucleic-acid parts orfragments of a nucleic acid as contemplated herein, can be introducedinto a host cell such that the resulting host cell contains a nucleicacid as contemplated herein or a vector as contemplated herein. Thisprocedure is particularly suitable if the host cell already contains oneor more components of a nucleic acid as contemplated herein or of avector as contemplated herein, and the additional components are thenadded accordingly. Methods for transforming cells are established in theprior art and are sufficiently known to a person skilled in the art. Inprinciple, all cells, i.e. prokaryotic or eukaryotic cells, are suitableas host cells. Preferred host cells are those which may beadvantageously managed genetically, which involves, for example,transformation using the nucleic acid or the vector and stableestablishment thereof, for example unicellular fungi or bacteria. Inaddition, preferred host cells are distinguished by good microbiologicaland biotechnological manageability. This relates, for example, to easeof culturing, high growth rates, low demands on fermentation media, andgood production and secretion rates for foreign proteins. Preferred hostcells as contemplated herein secrete the (transgenically) expressedprotein into the medium surrounding the host cells. Furthermore, theproteases can be modified, following preparation, by the cells thatproduced them, for example by the attachment of sugar molecules, byformylations, by aminations, etc. Post-translational modifications ofthis kind can influence the protease in terms of its function.

Those host cells of which the activity can be regulated due to geneticregulation elements which are provided on the vector, for example, butwhich may also be present in these cells from the outset, representother preferred embodiments. These host cells may be induced to express,for example by the controlled addition of chemical compounds which areused as activators, by changing the culturing conditions, or uponreaching a particular cell density. This provides for cost-effectiveproduction of the proteins as contemplated herein. An example of acompound of this kind is IPTG, as described above.

Prokaryotic or bacterial cells are preferred host cells. Bacteria aredistinguished by short generation times and low demands on the culturingconditions. Cost-effective culturing methods or preparation methods canthereby be established. Furthermore, a person skilled in the art has avast pool of experience with regard to bacteria in fermentationtechnology. Gram-negative or gram-positive bacteria may be suitable forspecific production for a wide variety of reasons, which should bedetermined by experiment in any given case, for example nutrientsources, product formation rate, time constraints, etc.

In the case of gram-negative bacteria, such as Escherichia coli,numerous proteins are secreted into the periplasmatic space, i.e. thecompartment between the two membranes which enclose the cells. This maybe advantageous for specific applications. Furthermore, gram-negativebacteria may also be formed such that they secrete the expressedproteins not only into the periplasmatic space, but also into the mediumsurrounding the bacterium. By contrast, gram-positive bacteria, forexample Bacilli or actinomycetes or other representatives of theactinomycetales, have no outer membrane, and therefore secreted proteinsare released directly into the medium surrounding the bacteria,generally the nutrient medium, from which the expressed proteins may bepurified. They may be isolated directly from the medium or processedfurther. Moreover, gram-positive bacteria are related to or identical tomost origin organisms for industrially significant enzymes and theythemselves usually form comparable enzymes, such that they have asimilar codon usage and the protein synthesis apparatus thereof isnaturally aligned accordingly.

Host cells as contemplated herein may be altered in terms of theirrequirements for culture conditions, may have different or additionalselection markers, or may express different or additional proteins.These host cells may be in particular host cells that express aplurality of proteins or enzymes transgenically.

The present disclosure can be used, in principle, for allmicroorganisms, in particular for all fermentable microorganisms,particularly preferably for those from the Bacillus genus, and leads toit being possible to prepare proteins as contemplated herein by usingmicroorganisms of this kind. Microorganisms of this kind then constitutehost cells within the meaning of the present disclosure.

In a further embodiment of the present disclosure, the host cell is abacterium, preferably a bacterium selected from the group of the generaof Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium,Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, morepreferably a bacterium selected from the group of Escherichia coli,Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillusglobigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans,Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum,Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor andStenotrophomonas maltophilia.

However, the host cell may also be a eukaryotic cell which has anucleus. Therefore, the present disclosure further relates to a hostcell which has a nucleus. Unlike prokaryotic cells, eukaryotic cells areable to modify the formed protein post-translationally. Examples ofeukaryotic cells are fungi such as actinomycetes or yeasts such asSaccharomyces or Kluyveromyces. This may be particularly advantageous,for example, if, in the context of their synthesis, the proteins areintended to undergo specific modifications which systems of this kindallow. The modifications which are carried out by eukaryotic systems,particularly in the context of protein synthesis, include, for example,the binding of low-molecular-weight compounds such as membrane anchorsor oligosaccharides. Oligosaccharide modifications of this kind may bedesirable, for example, as a means to reduce the allergenicity of anexpressed protein. A co-expression with the enzymes formed naturally bycells of this kind, such as cellulases, can also be advantageous.Furthermore, thermophilic fungal expression systems, for example, may beparticularly suitable for expressing temperature-resistant proteins orvariants.

The host cells as contemplated herein are cultured and fermented in aconventional manner, for example in batch or continuous systems. In thefirst case, a suitable nutrient medium is inoculated with the hostcells, and the product is harvested from the medium after a period oftime that can be determined by experiment. Continuous fermentation isdistinguished by the achievement of a steady state in which, over acomparatively long period of time, some cells die, but also regenerate,and at the same time, the formed protein can be removed from the medium.

Host cells as contemplated herein are preferably used in order toprepare proteases as contemplated herein. Therefore, the presentdisclosure also relates to a method for preparing a protease,comprising:

a) culturing a host cell as contemplated herein, and

b) isolating the protease from the culture medium or from the host cell.

This subject of the present disclosure preferably includes fermentationmethods. Fermentation methods are known per se from the prior art, andconstitute the actual large-scale production step, generally followed bya suitable method for purifying the prepared product, for example theproteases as contemplated herein. All fermentation methods which arebased on a corresponding method for preparing a protease as contemplatedherein constitute embodiments of this subject of the present disclosure.

Fermentation methods which are exemplified in that the fermentation iscarried out via an inflow strategy are in particular considered. Here,the media components that are consumed by the continuous culturing arefed in. Significant increases both in the cell density and in the cellmass or dry mass, and/or in particular in the activity of the proteaseof interest, can be achieved in this way. Furthermore, the fermentationmay also be designed in such a way that undesirable metabolic productsare filtered out, or neutralized by adding a buffer, or appropriatecounterions.

The prepared protease can be harvested from the fermentation medium. Afermentation method of this kind is preferred over isolation of theprotease from the host cell, i.e. product recovery from the cell mass(dry mass); however, said method requires that suitable host cells orone or more suitable secretion markers or mechanisms and/or transportsystems be provided, so that the host cells secrete the protease intothe fermentation medium. Alternatively, without secretion, the proteasecan be isolated from the host cell, i.e. separated from the cell mass,for example by precipitation with ammonium sulfate or ethanol, or bychromatographic purification.

All aforementioned elements can be combined to form methods forpreparing proteases as contemplated herein.

The present disclosure also relates to an agent which contains aprotease as contemplated herein, as described above. The agent ispreferably a washing or cleaning agent.

This covers all conceivable types of washing or cleaning agents,including both concentrates and agents to be used in undiluted form, foruse on a commercial scale in washing machines or for washing or cleaningby hand. These agents include, for example, washing agents for textiles,carpets or natural fibers for which the term “washing agent” is used.These also include, for example, dishwashing detergents for dishwashersor manual dishwashing detergents or cleaners for hard surfaces, such asmetal, glass, porcelain, ceramics, tiles, stone, coated surfaces,plastics materials, wood or leather for which the term “cleaning agent”is used, i.e. in addition to manual and automatic dishwashingdetergents, also abrasive cleaners, glass cleaners, WC rimblocks, etc.Within the scope of the present disclosure, the washing and cleaningagents also include auxiliary washing agents, which are added to theactual washing agent when washing textiles manually or using a machinein order to achieve an additional effect. Furthermore, within the scopeof the present disclosure, washing and cleaning agents also includetextile pre-treatment and post-treatment agents, i.e. agents with whichthe piece of laundry comes into contact before it is actually washed,for example in order to loosen stubborn dirt, and also agents whichimpart other desirable properties to the laundry, for example softnessto touch, crease resistance or low static charge, in a step that comesafter the actual textile washing process. The agents mentioned lastinclude, inter alia, softeners.

The washing or cleaning agents as contemplated herein, which may bepresent in the form of powdered solids, compressed particles,homogeneous solutions or suspensions, can contain, in addition to aprotease as contemplated herein, all known ingredients that are commonin agents of this kind, at least one further ingredient preferably beingpresent in the agent. The agents as contemplated herein may containsurfactants, builders, bleaching agents, such as peroxygen compounds inparticular, and/or bleach activators. They may also containwater-miscible organic solvents, further enzymes, sequestering agents,electrolytes, pH regulators and/or further auxiliaries such as opticalbrighteners, graying inhibitors, foam regulators, and dyes andfragrances, and combinations thereof.

In particular, a combination of a protease as contemplated herein withone or more further ingredient(s) of the agent is advantageous, since anagent of this kind has improved cleaning performance in preferredembodiments as contemplated herein on account of synergies obtainedthereby. In particular, such synergy can be achieved by the combinationof a protease as contemplated herein with a surfactant and/or a builderand/or a peroxygen compound and/or a bleach activator. However, inpreferred embodiments, the agent as contemplated herein may not containboric acid.

Advantageous ingredients of agents as contemplated herein are disclosedin international patent application WO2009/121725, starting on thepenultimate paragraph of page 5 and ending on page 13 after the secondparagraph. Reference is made explicitly to this disclosure and thecontent thereof is incorporated in the present patent application.

An agent as contemplated herein preferably contains the protease in anamount of from about 2 μg to about 20 mg, preferably from about 5 μg toabout 17.5 mg, particularly preferably from about 20 μg to about 15 mg,and very particularly preferably from about 50 μg to about 10 mg per gof the agent. Furthermore, the protease contained in the agent and/orfurther ingredients of the agent may be encapsulated in a substance thatis impermeable to the enzyme at room temperature or in the absence ofwater, which substance becomes permeable to the enzyme under useconditions of the agent. Such an embodiment of the present disclosure isthus exemplified in that the protease is encapsulated in a substancethat is impermeable to the protease at room temperature or in theabsence of water. Furthermore, the washing or cleaning agent itself canalso be packaged in a container, preferably an airtight container, fromwhich it is released shortly before use or during the washing process.

In other embodiments of the present disclosure, the agent

a) is present in solid form, in particular as a flowable powder having abulk density of from about 300 g/l to about 1200 g/l, in particular fromabout 500 g/l to about 900 g/l, or

b) is present in paste or liquid form, and/or

c) is present in gel or pouch form, and/or

d) is present as a single-component system, or

e) is divided into a plurality of components.

These embodiments of the present disclosure cover all solid, powder,liquid, gel or paste dosage forms of agents as contemplated herein thatmay optionally also consist of a plurality of phases, and may be presentin compressed or uncompressed form. The agent may be present in the formof a flowable powder, in particular having a bulk density of from about300 g/l to about 1200 g/l, more particularly from about 500 g/l to about900 g/l or from about 600 g/l to about 850 g/l. The solid dosage formsof the agent also include extrudates, granules, tablets or pouches.Alternatively, the agent may also be a liquid, gel or paste, for examplein the form of a non-aqueous liquid washing agent or a non-aqueous pasteor in the form of an aqueous liquid washing agent or water-containingpaste. Furthermore, the agent may be present as a single-componentsystem. Agents of this kind consist of one phase. Alternatively, anagent can also consist of a plurality of phases. An agent of this kindis therefore divided into a plurality of components.

Washing or cleaning agents as contemplated herein may only contain aprotease. Alternatively, they may also contain further hydrolyticenzymes or other enzymes in a concentration that is expedient in termsof the effectiveness of the agent. Another embodiment of the presentdisclosure thus relates to agents which also comprise one or morefurther enzymes. All enzymes which can develop catalytic activity in theagent as contemplated herein, in particular a lipase, amylase,cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase,xyloglucanase, β-glucosidase, pectinase, carrageenase, perhydrolase,oxidase, oxidoreductase or another protease that is different from theprotease as contemplated herein, and mixtures thereof, can preferably beused as further enzymes. Further enzymes are contained in the agentadvantageously in an amount of from about 1×10⁻⁸ to about 5 wt. % ineach case, based on the active protein. Each further enzyme is containedin agents as contemplated herein in an amount of, in order of increasingpreference, from about 1×10⁻⁷ to about 3 wt. %, from about 0.00001 toabout 1 wt. %, from about 0.00005 to about 0.5 wt. %, from about 0.0001to about 0.1 wt. %, and most particularly preferably from about 0.0001to about 0.05 wt. %, based on the active protein. The enzymesparticularly preferably have synergistic cleaning performances withrespect to particular stains or marks, i.e. the enzymes contained in theagent composition assist one another in terms of the cleaningperformance thereof. Very particularly preferably, such synergy existsbetween the protease contained as contemplated herein and a furtherenzyme of an agent as contemplated herein, in particular between thestated protease and an amylase and/or a lipase and/or a mannanase and/ora cellulase and/or a pectinase. Synergistic effects can occur not onlybetween different enzymes, but also between one or more enzymes andother ingredients of the agent as contemplated herein.

The present disclosure further relates to a method for cleaning textilesor hard surfaces, exemplified in that an agent as contemplated herein isused in at least one method step, or in that a protease as contemplatedherein becomes catalytically active in at least one method step, inparticular such that the protease is used in an amount of from about 40μg to about 4 g, preferably from about 50 μg to about 3 g, particularlypreferably from about 100 μg to about 2 g, and very particularlypreferably from about 200 μg to about 1 g.

In various embodiments, the above-described method is distinguished inthat the protease is used at a temperature of from about 0 to about 100°C., preferably from about 0 to about 60° C., more preferably from about20 to about 45° C., and most preferably at a temperature of about 40° C.

These embodiments include both manual and automatic methods, automaticmethods being preferred. Methods for cleaning textiles are generallydistinguished in that various substances that have a cleaning effect areapplied to the item to be cleaned in a plurality of method steps andwashed off after the contact time, or in that the item to be cleaned istreated with a washing agent or a solution or dilution of this agent insome other way. The same applies to methods for cleaning all materialsother than textiles, in particular hard surfaces. All conceivablewashing or cleaning methods can be enhanced in at least one of themethod steps by the use of a washing or cleaning agent as contemplatedherein or a protease as contemplated herein, and then constituteembodiments of the present disclosure. All elements, subjects andembodiments that are described for proteases as contemplated herein andagents that contain them can also be applied to this subject of thepresent disclosure. Therefore, at this juncture, reference is explicitlymade to the disclosure at the corresponding point when it was indicatedthat this disclosure also applies to the above methods as contemplatedherein.

Since proteases as contemplated herein naturally already have hydrolyticactivity and these also develop in media that otherwise have no cleaningforce, such as in simple buffers, an individual and/or the only step ofa method of this kind can consist in bringing a protease as contemplatedherein into contact with the stain as the only component that has acleaning effect, preferably in a buffer solution or in water. Thisconstitutes a further embodiment of this subject of the presentdisclosure.

Methods for treating textile raw materials or for textile care in whicha protease as contemplated herein becomes active in at least one methodstep also constitute alternative embodiments of this subject of thepresent disclosure. Of such methods, methods for textile raw materials,fibers or textiles having natural components are preferred, and veryparticularly for those containing wool or silk.

Finally, the present disclosure further relates to the use of theproteases described herein in washing or cleaning agents, for example asdescribed above, for (improved) removal of protein-containing stains,for example from textiles or hard surfaces.

All elements, subjects and embodiments that are described for proteasesas contemplated herein and agents that contain them can also be appliedto this subject of the present disclosure. Therefore, at this juncture,reference is explicitly made to the disclosure at the correspondingpoint when it was indicated that this disclosure also applies to theabove use as contemplated herein.

EXAMPLES Example 1: Washing Test

Overview of the mutations:

This present disclosure relates to an alkaline subtilisin-type proteasefrom Bacillus pumilus. Variants were produced from this protease byemploying random mutagenesis, which variants were then analyzed, interalia, for improved washing performance.

Variant Sequence SEQ ID NO: Mutant 1 P86S S236A 2 Mutant 2 Q62E 3 Mutant3 Q62E L82F 4 Mutant 4 Q10E 5 Mutant 5 N130D T141K T253S 6 Mutant 6 P86A7 Mutant 7 P9H Q62E L82F 8 Mutant 8 Q62E L82F N187H 9 Mutant 9 Q62EN130D 10 Mutant 10 P86S N187H S236A 11

Washing Agent Matrix Used

This is a commercially available washing agent matrix which was used forthe washing test:

wt. % active wt. % active substance substance in the raw in the Chemicalname material formulation demineralized water 100 remainder alkylbenzenesulfonic acid 96 4.4 anionic surfactants 70 5.6 C12-C18 fatty acid Nasalt 30 2.4 non-ionic surfactants 100 4.4 phosphonates 40 0.2 citricacid 100 1.4 NaOH 50 0.95 defoamers t.q. 0.01 glycerol 100 2preservatives 100 0.08 ethanol 93 1 without optical brighteners,perfume, dye and enzymes. Dosage 4.7 g/L

Protease Activity Assays

The activity of the protease is determined by releasing the chromophorepara-nitroaniline from the substrate succinylalanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; BachemL-1400). The release of pNA causes an increase in the extinction at 410nm, the temporal progression of which is a measure of enzymaticactivity.

The measurement is taken at a temperature of 25° C., a pH of 8.6, and awavelength of 410 nm. The measurement time is 5 minutes, with ameasurement interval of from 20 to 60 seconds.

Measurement formulation:

10 μL AAPF solution (70 mg/mL)

1000 μL tris/HCl (0.1 M; pH 8.6 with 0.1% Brij 35)

10 μL diluted protease solution

Kinetics produced over 5 minutes at 25° C. (410 nm).

Washing Test and Results

The mini washing test was carried out using Bacillus subtilis culturesupernatants that contained the analyzed protease mutants as a result ofheterologous expression. The supernatants were used such that they hadthe same activity as the benchmark (wild-type protease) at aconcentration that is customary in the market for proteases in washingagents.

Conditions: 40° C., 16° dH water, 1 h

Stains:

1. CFT CS038 (egg yolk with pigment (dried) on cotton; Center forTestmaterials Cft B.V., Vlaardingen, NL)

2. CFT PC-10 (peanut oil, pigment, milk on polyester/cotton; Center forTestmaterials Cft B.V., Vlaardingen, NL)

3. WfK 10N (egg/pigment on cotton, wfk-Cleaning Technology Institutee.V., Krefeld, DE)

4. CFT C-03 (cocoa/carbon black on cotton; Center for Testmaterials CftB.V., Vlaardingen, NL)

5. EMPA 112 (cocoa on cotton; Swissatest Testmaterialien AG, St. Gallen,CH)

6. CFT C-05 (blood, milk, ink on cotton; Center for Testmaterials CftB.V., Vlaardingen, NL)

A woven fabric blank (diameter=10 mm) was placed in a microtiter plate,washing liquor was preheated to 40° C., the end concentration was 4.7g/L, the liquor and enzyme were put on the stain and incubated for 1 hat 40° C. and 600 rpm, then the stain was rinsed several times withclear water and left to dry, and the lightness was determined using acolor measurement device. The lighter the woven fabric, the better thecleaning performance. What was measured in this case was the Lvalue=lightness, and the higher the value the lighter the stain. The sumof the six stains in %, based on the wild type, is given below.

Washing performance (delta L) (based on the washing performance of theVariant WT protease (SEQ ID NO: 1)) Mutant 1 142% Mutant 2 146% Mutant 3142% Mutant 4 162% Mutant 5 148% Mutant 6 167% Mutant 7 156% Mutant 8129% Mutant 9 112% Mutant 10 121%

While at least one exemplary embodiment has been presented in theforegoing detailed description, it should be appreciated that a vastnumber of variations exist. It should also be appreciated that theexemplary embodiment or exemplary embodiments are only examples, and arenot intended to limit the scope, applicability, or configuration of thevarious embodiments in any way. Rather, the foregoing detaileddescription will provide those skilled in the art with a convenient roadmap for implementing an exemplary embodiment as contemplated herein. Itbeing understood that various changes may be made in the function andarrangement of elements described in an exemplary embodiment withoutdeparting from the scope of the various embodiments as set forth in theappended claims.

The invention claimed is:
 1. A protease comprising an amino acidsequence that has at least 92.5% sequence identity to the amino acidsequence set forth in SEQ ID NO:2 over the entire length thereof, andthat has an amino acid substitution at one or more of positions P9, Q10,Q62, L82, P86, N130, T141, N187, S236, and T253, based on the numberingaccording to SEQ ID NO:2, wherein the amino acid sequence that has theat least 92.5% sequence identity to the amino acid sequence set forth inSEQ ID NO:2 has protease activity, and wherein the at least one aminoacid substitution is selected from the group of P9H, Q10E, Q62E, L82F,P86S, P86A, N130D, T141K, N187H, S236A, T253S, and combinations thereof,based on the numbering according to SEQ ID NO:2.
 2. The proteaseaccording to claim 1, wherein the protease comprises one of thefollowing sets of amino acid substitutions: (i) P86S and S236A; (ii)Q62E; (iii) Q62E and L82F; (iv) Q10E; (v) N130D, T141K and T253S; (vi)P86A; (vii) P9H, Q62E and L82F; (viii) Q62E, L82F and N187H; (ix) Q62Eand N130D; or (x) P86S, N187H and S236A.
 3. The protease according toclaim 1, wherein the protease is utilized in a washing or cleaning agentfor removing peptide-containing or protein-containing stains.
 4. Theprotease according to claim 1, wherein the protease comprises the aminoacid substitutions P86S and S236A based on the numbering according toSEQ ID NO:2.
 5. The protease according to claim 1, wherein the proteasecomprises the amino acid substitution Q62E based on the numberingaccording to SEQ ID NO:2.
 6. The protease according to claim 1, whereinthe protease comprises the amino acid substitutions Q62E and L82F basedon the numbering according to SEQ ID NO:2.
 7. The protease according toclaim 1, wherein the protease comprises the amino acid substitution Q10Ebased on the numbering according to SEQ ID NO:2.
 8. The proteaseaccording to claim 1, wherein the protease comprises the amino acidsubstitutions N130D, T141K and T253S based on the numbering according toSEQ ID NO:2.
 9. The protease according to claim 1, wherein the proteasecomprises the amino acid substitution P86A based on the numberingaccording to SEQ ID NO:2.
 10. The protease according to claim 1, whereinthe protease comprises the amino acid substitutions P9H, Q62E and L82Fbased on the numbering according to SEQ ID NO:2.
 11. The proteaseaccording to claim 1, wherein the protease comprises the amino acidsubstitutions Q62E, L82F and N187H based on the numbering according toSEQ ID NO:2.
 12. The protease according to claim 1, wherein the proteasecomprises the amino acid substitutions Q62E and N130D based on thenumbering according to SEQ ID NO:2.
 13. The protease according to claim1, wherein the protease comprises the amino acid substitutions P86S,N187H and S236A based on the numbering according to SEQ ID NO:2.
 14. Theprotease according to claim 1, wherein the protease comprises an aminoacid sequence that has at least 95% sequence identity to the amino acidsequence set forth in SEQ ID NO:2 over the entire length thereof. 15.The protease according to claim 1, wherein the protease comprises anamino acid sequence that has at least 97% sequence identity to the aminoacid sequence set forth in SEQ ID NO:2 over the entire length thereof.